Batagur affinis (Southern river terrapin or locally known as "tuntung sungai") in the Indochina region is a critically endangered freshwater turtle species. Despite numerous fundamental studies on the biological properties of B. affinis, there are significant concerns regarding the extent of population differentiation, genomic structure, and genetic variability within their populations due to a lack of genetic-based investigations. A lack of basic circumstances such as phylogenetic relationships and population genetic structure would harm their long-term conservation. As a result, this study was carried out for the first time to characterise the genetic structure of two B. affinis subspecies (B. affinis affinis and B. affinis edwardmolli) using cross-amplification of Batagur trivittata microsatellites and a total of 80 collected specimens from four Malaysian populations. Each sample's genomic DNA was taken for polymerase chain reaction (PCR) amplification and fragment analysis. Five microsatellite primers were used to identify polymorphic loci in B.affinis samples. In B. affinis, the number of alleles per locus ranged from 21 to 37. Microsatellites analysis revealed that there was little genetic heterogeneity among the B. affinis populations. The average observed heterozygozygosity (HO = 0.51) was lower than the typical heterozygozygosity found in most aquatic populations (HO = 0.79). The presence of inbreeding across distinct populations of B. affinis was also supported by the high FIS values (mean FIS = 0.3850) and low FST values (mean FST = 0.0893). Finally, this study offered light on the population structure of B.affinis in the Indochina region.